Chromogenic enzyme substrates

ABSTRACT

This invention provides novel chromogenic enzyme substrates which are indoxyl β-D-ribofuranosides. A process for their production is provided. Methods for detecting β-D-ribofuranosidase activity are given. The advantages of these novel compounds includes: detecting β-D-ribofuranosidase activity with high sensitivity and low substrate concentrations and use with other enzyme indicators in situations where a plurality of enzyme activities is to be visualized simultaneously e.g. for identifying bacteria growth on solid growth media. The synthesis and use of 5-bromo-4-chloro-3-indolyl-β-D-ribofuranoside is exemplified.

This invention relates to chromogenic enzyme substrates.

Indicator enzyme substrates comprise an enzyme cleavable portion (eg amonosaccharyl) and a portion which forms a detectable indicator oncleavage (eg a chromogenic or fluorogenic group). A large number ofglycoside-based enzyme substrates are known and are used extensively inmicrobiology, molecular biology and other fields. Glycosides of manydifferent carbohydrates have been synthesised and utilised for detectionpurposes. The enzymes that are detected by these glycosides are oftengroup specific (i.e. show relatively little specificity towards oneportion of the substrate upon which they act) and therefore a widevariety of aglycones (i.e. indicator portions) can be tolerated. Thus,in the case of β-galactosidase many different β-galactosides have beenused in the detection of it. Examples include o-nitrophenyl-,p-nitrophenyl-, indoxyls-(5-bromo-4-chloro-3-indolyl and6-chloro-3-indolyl), 4-methylumbelliferyl-, 2-naphthyl-,6-bromo-2-naphthyl-, cyclohexenoesculetin-(CHE), alizarin-,naphthol-ASBI- and phenyl-β-D-galactosides. Glycosides containingglucuronic acid, glucose, galactose, mannose, fucose, arabinose,N-acetylglucosamine, N-acetylgalactosamine, sialic acid, xylose, andcellobiose carbohydrate moieties are amongst those most frequentlyencountered in enzyme substrate applications. Many of these, such asβ-D-glucuronides, α- and β-D-galactopyranosides and α- andβ-D-glucopyranosides have found widespread use in the identification andenumeration of bacteria in areas such as clinical, food, veterinary,environmental and water microbiology. At the present time there arenumerous commercial media and test kits available containing enzymesubstrates, which show the presence of bacteria, yeast and othermicro-organisms by the generation of coloured colonies or solutions.

Co-pending PCT application based on British Application 0125528.0describes the synthesis of certain β-D-ribofuranoside derivatives aschromogenic substrates for the detection of β-D-ribofuranosidaseactivity through the generation of insoluble coloured precipitates thatare formed from the aglycone portion after enzymatic cleavage.β-D-Ribofuranosidase activity is an enzyme activity capable of cleavingβ-D-ribofuranosyl groups. GB 0125528.0 illustrates the utility ofdetecting β-D-ribofuranosidase activity in diagnostic microbiology. Onefeature of many of the exemplified molecules described in GB 0125528.0is that the aglycones are derivatives of catechol. These substrates maybe conveniently used in solid media such as agar-based media. Byinclusion of an iron salt such as ferric ammonium citrate into themedium, these substrates produce brown or black insoluble precipitatesthat clearly indicate the site of enzymatic activity. This is useful indistinguishing microbes or other entities which possessβ-D-ribofuranosidase activity from those which do not. A disadvantage ofthe very intense dark brown or black precipitate produced by substratessuch as 3′,4′-dihydroxyflavone-4′-β-D-ribofuranoside when used atdiagnostically adequate concentrations is that it can mask the colourproduced by another chromogenic substrate incorporated into the samemedium for the detection of a different enzyme activity. It may readilybe appreciated by those skilled in the art that under certaincircumstances this is a disadvantage when trying to develop usefuldiagnostic media which depend on the visualisation of other enzymeactivities in addition to, and simultaneously with, β-D-ribofuranosidaseactivity.

Indoxyl-glycosides of various monosaccharides have been reported and arereadily available commercially. For example WO-A-99/50438 describesvarious indoxyl-glycosides including N-methyl-indoxyl-glycosides.Indoxyl-glycosides are well established as chromogenic enzyme substratesand they have found wide application in diagnostic microbiologyincluding their use in solid media [M. Manafi, Int. J. Food Microbiol.,31, 45, (1996)]. These substrates yield insoluble coloured precipitatesafter enzymatic cleavage. The coloured precipitate is derived mainlyfrom two molecules of the cleaved aglycone, indoxyl, which combine viaan oxidative process to form an indigo dye [S. Cotson and S. J. Holt,Proc. Roy. Soc. B, 148, 506, (1958)].

Although the precise observed colours of the insoluble precipitates mayvary slightly depending on the exact conditions under which they areproduced and may be influenced by, for instance, other componentspresent in the media, the colour of the precipitate given by theprincipal indoxyl moieties commonly used in diagnostic microbiology maybe described approximately as follows;

Indoxyl Colour 5-Bromo-4-chloroindoxyl Green 5-Bromoindoxyl Dark blueIndoxyl Blue 5-Bromo-6-chloroindoxyl Magenta 6-Chloroindoxyl Rose

By choosing colours of these precipitates which are sufficientlycontrasting, it is possible to design diagnostic media which detect twoenzyme activities simultaneously by using two or more differentindoxyl-substrates in which both the indoxyl and the carbohydrate orenzyme cleavable moieties are different in each substrate. Media of thisnature have been described [J. N. Roth and W. J. Ferguson, U.S. Pat. No.5,210,022 (1993); D. G. Flowers and M. Sternfeld, U.S. Pat. No.5,364,767 (1994)]. One feature of interest with media containingdifferent indoxyl substrates is that combinations of colours may beformed when two of the sought after enzyme activities are present in thesame entity, and this can help to achieve a more selective detectionsystem for the microbes under investigation.

The prior art contains several examples of the chemical synthesis ofindoxyl-glycosides. All of these reported examples employ peracylatedsugar halides as the glycosyl donor or, in the case of glucuronides, theequivalent methyl triacetylglucuronyl bromide [K. Yoshida et al., Anal.Biochem., 58, 77, (1974); K. Yoshida et al, Chem. Pharm. Bull., 32,1759, (1975); A. N. Ley et al., Can. J. Microbiol., 34, 690, (1988); S:Wolfe and R. J. Bowers, EP-A-0284725A2, (1988)]. The glycosyl acceptoris either a 1-acetylindoxyl derivative of the general formula (I) or, inone example, 3-acetyl-5-bromo-4-chloroindoxyl

Most of these methods are base-promoted glycosylations and may thereforebe considered as belonging to the classic Michael-type glycosylation(reaction in alcohol) or its variation as developed by Mannich (reactionin acetone/water). The first synthesis of an indoxyl-glycoside, indoxylβ-D-glucopyranoside (Plant Indican) was reported by Robertson in 1927[A. Robertson, J. Chem. Soc, 1937, (1927)] using Mannich-typeconditions. He succeeded in condensing 1-acetylindoxyl (I, V═H) withacetobromoglucose by use of potassium hydroxide in aqueous acetone tomake the peracetylated indoxyl-glucoside intermediate. De-acetylationfurnished the required indoxyl β-D-glucopyranoside. However, hepreferred another route to the peracetylated intermediate, citing thethen difficulty in preparing 1-acetylindoxyl as one reason for this. Hisalternative route involved condensing methyl indoxyl-2-carboxylate (I,V¹⁻³═H, V⁴═CO₂Me) with acetobromoglucose, also by the aid of potassiumhydroxide in aqueous acetone. However, several further steps wererequired before the desired final product could be obtained because ofthe need to de-carboxylate the indole nucleus at position-2, a processinvolving the forcing conditions of heating with sodium acetate inacetic anhydride at 160° C. The route involving the coupling of aprotected sugar halide with the the appropriate methylindoxyl-2-carboxylate has had little further application [A. Robertsonand R. B. Waters, J. Chem. Soc., 72, (1931); Ley et al., loc. cit; S.Wolfe and R. J. Bowers, loc. cit.]. The more expeditious route providedby the direct coupling of 1-acetylindoxyls with a fully acylated sugarhalide (most often the acetobromosugar) has been the usual method ofchoice. This avoids the need for a de-carboxylation step. The necessary1-acetylindoxyls (1, V⁴═H) are very readily obtained by the methodsdeveloped by Holt and Sadler [S. J. Holt et al., J. Chem. Soc., 1217,(1958); cf. S. J. Holt and P. W. Sadler, Proc. Roy. Soc. B, 148, 481,(1958)]. After coupling 1-acetylindoxyls with a peracylated sugarhalide, Zemplén-type deprotection (sodium methoxide in methanol) yieldsthe end product indoxyl glycoside. This approach was chosen by Andersonand Leaback [F. B. Anderson and D. Leaback, Tetrahedron, 12, 236,(1961)] for the synthesis of three 5-bromo-3-indolyl-based glycosides.In one example of theirs, 1-acetyl-5-bromoindoxyl (1, V²═Br, V¹, V³,V⁴═H) was coupled with acetobromogalactose via the use of sodiumhydroxide in aqueous acetone. After recovery of the peracetylatedintermediate, the final product was obtained directly afterde-protection with methanolic sodium methoxide and work-up. Thesynthesis of a 5-bromo-3-indolyl disaccharide from the acetobromosugarvia the conditions of Anderson and Leaback has been reported recently[S. Kaneko et al., Biosci. Biotechnol. Biochem., 64, 741, (2000)]. Therange of halogenated indoxyl-monosaccharides was further extended byHorwitz and co-workers [J. P. Horwitz et al., J. Med. Chem., 7, 574,(1964)]. In one synthesis by these investigators,1-acetyl-5-bromo-4-chloroindoxyl (X—OH) (I, V¹═Cl, V²═Br, V³⁻⁴═H) wascoupled with acetobromogalactose under the conditions used by Andersonand Leaback. After de-protection of the peracetylatedindoxyl-galactoside they obtained 5-bromo-4-chloro-3-indolylβ-D-galactopyranoside (X-Gal). X-Gal is currently the most widelyencountered indoxyl-glycoside used in diagnostic microbiology as well asin other fields such as molecular biology. In the same paper, Horwitzand co-workers also employed 3-acetyl-5-bromo-4-chloroindoxyl [S. J.Holt and P. W. Sadler, ioc. cit.] as an intermediate. Using thisreactant it was necessary to remove the 3-acetyl group with sodium inmethanol to generate the indoxyl sodium salt in situ prior to treatingit with acetobromoglucose. Because the conditions of the reaction alsoled to the removal of all the other acetyl protecting groups, the fullydeprotected glycoside, 5-bromo-4-chloro-3-indolyl β-D-glucopyranoside(X-glucoside) was furnished in a single step. However this route hasthree distinct disadvantages. Firstly, 3-acetylindoxyls are moredifficult to prepare than 1-acetylindoxyls. Secondly, their much reducedstability makes them more difficult to work with. Thirdly, because thereaction is “one-pot” and the yield is low, there is a substantialquantity of highly coloured residue which has to be removed before theproduct can be usefully employed as an enzyme substrate, and this isvery cumbersome to accomplish satisfactorily on a large preparativescale.

More recently, Berlin and Sauer [W. Berlin and B. Sauer, Anal. Biochem.,243, 171, (1996)] reported difficulty with the base promoted reactionbetween 1-acetyl-5-bromoindoxyl (I, V²═Br, V¹, V³, V⁴═H) and aperbenzoyl-arabinofuranosyl bromide. However, they were able to obtain asuccessful glycosylation reaction by using silver triflate as thecatalyst in dichloromethane. These conditions represent a variation ofthe classic Koenigs-Knorr reaction [K. Toshima and K. Tatsuta, Chem.Rev., 93, 1503, (1993)].

Before the present invention, indoxyl derivatives of β-D-ribofuranosideshad not been described.

A first aspect of this invention provides an indoxyl β-D-ribofuranoside,of the formula II

wherein R¹⁻⁴ are independently H, halide, nitro or C₁₋₆alkyl groups andR⁵ is H, C₁₋₆alkyl, or aralkyl or a substituted derivative or ester ofthe indoxyl β-D-ribofuranoside.

A second aspect of this invention provides a process for producing anindoxyl β-D-ribofuranoside according to the first aspect of thisinvention; comprising a) contacting a protectedβ-D-ribofuranosyltrichloroacetimidate with a compound of formula III

wherein R¹⁻⁴ are independently H, halide, nitro or C₁₋₆alkyl groups andT⁵ is, acyl, trialkylsilyl or other protecting groups in the presence ofa catalyst to form a protected indoxyl-β-D-ribofuranoside; and b)removing the protecting groups.

Protecting groups are moieties attached to reactive hydroxyl groups onβ-D-ribofuranosyl or the indoxyl nitrogen to prevent reactions occurringwhich are not required. The most commonly used protecting group isacetyl.

A third aspect of this invention provides a method for detectingβ-D-ribofuranosidase activity in a sample comprising;

a) contacting the sample with an indoxyl β-D-ribofuranoside according tothe first aspect of this invention; wherein said indoxylβ-D-ribofuranoside comprises a β-D-ribofuranosyl moiety and an indoxylmoiety, said β-D-ribofuranosyl moiety being cleavable byβ-D-ribofuranosidase from the indoxyl moiety releasing the indoxylmoiety which forms a coloured compound; and

b) concluding whether β-D-ribofuranosidase activity is present bydetecting whether a coloured compound is formed from the indoxyl moiety.

According to a fourth aspect of this invention a kit is providedcomprising

a) an indoxyl β-D-ribofuranoside according to the first aspect of thisinvention and

b) a component for use in producing a microbial growth medium.

According to a fifth aspect of this invention a composition is providedwhich comprises an indoxyl β-D-ribofuranoside according to the firstaspect of this invention and another component.

Upon cleavage by β-D-ribofuranosidase, the indoxyl moiety of indoxylβ-D-ribofuranosides would produce a range of coloured compounds, asindicated above different from those given by the catechol-derivedβ-D-ribofuranosides exemplified in GB 0125528.0. Furthermore, unlikecompounds such as those formed from3′,4′-dihydroxyflavone-4′-β-D-ribofuranoside in a medium containing aniron salt, the coloured compound produced by indoxyl β-D-ribofuranosidesafter enzymatic cleavage is not likely to mask completely the colourgenerated by a different enzyme substrate, (such as the colouredcompound produced by a different indoxyl portion attached to anothersugar or enzyme cleavable group), incorporated into the medium for thevisualisation of a different enzyme activity. Moreover, it is envisagedthat some new chromogenic media of the present invention would enablethe presence of β-D-ribofuranosidase activity to be detected by the useof a chromogenic β-D-ribofuranoside which is masked by a second, morehighly coloured compound liberated by a different enzyme activity in thesame sample after its action upon a second chromogenic enzyme substrate.It will be appreciated by those skilled in the art that under certaincircumstances this represents an advantage when trying to develop usefuldiagnostic media which depend on the visualisation of other enzymeactivities in addition to β-D-ribofuranosidase activity. It wastherefore considered that the synthesis of indoxyl β-D-ribofuranosidesand their application as chromogenic enzyme substrates is complementaryto that of the catechol-based β-D-ribofuranosides exemplified in GB0125528.0 and therefore overcomes the present limitations in designingnovel chromogenic media in which the detection of β-D-ribofuranosidaseactivity is critical.

Indoxyl β-D-ribofuranosides of the current invention are defined byformula II above. It is generally preferred that positions R¹ to R⁴ areH, C₁₋₆ alkyl or halide and the halides are generally bromo or chloro.R⁵ can be C₁₋₆ alkyl or aralkyl such as benzyl, but is generally H ormethyl. Specially preferred indoxyl portions are selected from the groupconsisting of 5-bromo-4-chloro-3-indolyl (also called X),5-bromo-6-chloro-3-indolyl, 5-bromo-3-indolyl, 6-chloro-3-indolyl,3-indolyl, 4-chloro-3-indolyl, 6-bromo-3-indolyl, 6-fluoro-3-indolyl,5,7-dibromo-3-indolyl, 4,5-dichloro-3-indolyl, 5-nitro-3-indolyl,1-methyl-3-indolyl, 5-bromo-4-chloro-1-methyl-3-indolyl,5-bromo-6-chloro-1-methyl-3-indolyl, 5-bromo-1-methyl-3-indolyl,6-chloro-1-methyl-3-indolyl, 4-chloro-1-methyl-3-indolyl,6-bromo-1-methyl-3-indolyl, 6-fluoro-1-methyl-3-indolyl,5,7-dibromo-1-methyl-3-indolyl, 5,6-dibromo-3-indolyl,5,6-dibromo-1-methyl-3-indolyl and 5-nitro-1-methyl-3-indolyl and4,5-dichloro-1-methyl-3-indolyl.

The second aspect of this invention provides a process for producingindoxyl β-D-ribofuranosides and was developed after protracted andin-depth studies carried out by the inventor.

In order to provide compounds of the invention, it was attempted firstto try coupling 1-acetyl-5-bromo4-chloroindoxyl (X—OH)(1-acetyl-5-bromo4-chloroindol-3-ol) with acetobromo- oracetochlororibofuranose under the conditions used by Andersen andLeaback (loc. cit.) to make 5-bromo-3-indolyl glycosides and by Horwitzand co-workers (loc. cit.) to make X-Gal, the target molecule of thepresent invention therefore being 5-bromo-4-chloro-3-indolylβ-D-ribofuranoside (X-β-D-ribofuranoside). Thus, eitheracetobromoribofuranose or the somewhat more stableacetochlororibofuranose [H. Zinner, Chem. Ber., 83, 153, (1950)] (bothprepared from commercially available β-D-ribofuranose tetraacetate) andX—OH were treated with the appropriate quantity of sodium or potassiumhydroxide in aqueous acetone. Analysis of the reactions by tic (for theappearance of a new UV active product) and, where appropriate, byisolating any new products formed by column chromatography on silica gelC60 followed by nmr analysis, showed that none of the reactions producedthe expected protected indoxyl-ribofuranoside intermediate.

Ribosylation was then tried using the tribenzoyl-D-ribofuranosyl bromide[S. Hanessian and A. G. Pernet, Can. J. Chem., 52, 1280, (1974)] andchloride [H. M. Kissman et al., J. Amer. Chem. Soc., 77, 18, (1955)].Nonetheless, no product formation was observed in these reactions also.Owing to the failure to couple X—OH to a peracylated ribofuranosylhalide under the influence of either potassium or sodium hydroxide, analternative glycosylation procedure employing these halides was soughtThe Koenigs-Knorr reaction using protected sugar halides is a widelyused procedure to construct Olycosides and has already been applied inthe indoxyl-series [W. Berlin and B. Sauer, loc. cit.]. Originallydeveloped using silver salts as both the catalyst and the halideacceptor [W. Koenigs and E. Knorr, Chem. Ber., 34, 957, (1901)], thescope of the reaction has been extended over the years by the use ofother metal catalysts [K. Toshima and K. Tatsuta, loc. cit.]. Several ofthese were explored during work on the present invention with theaforementioned peracetyl- and perbenzoylribofuranosyl halides. Using thesolvents, acetonitrile, chloroform, dichloromethane, diethyl ether,dimethylformamide, nitromethane or pyridine, the following glycosylationcatalysts were tried, all without success; silver(I)carbonate,silver(I)cyanide, silver(I)oxide, silver(I)nitrate, silver(I)triflate,cadmium carbonate, mercury(II)bromide, mercury(II)chloride, andmercury(II)oxide.

The failure of the Koenigs-Knorr reaction to produce the desiredglycoside led to the assessment of other glycosylation methods notentailing the intermediary of a glycosyl halide in the key couplingstep. The use of 1-O-acylated glycosyl donors in glycosylations(Helferich reaction) was first demonstrated in 1933 [B. Helferich and E.Schmitz-Hillebrecht, Chem. Ber., 66, 378, (1933)]. This reaction ofteninvolves heating a peracylated sugar with an aglycone in the presence ofa Lewis acid promoter such as p-toluenesulfonic acid (PTSA), or zincchloride [E. M. Montgomery, N. K. Richtmyer and C. S. Hudson, J. Amer.Chem. Soc., 64, 690, (1942)]. Heating β-D-ribofuranose tetraacetate andX—OH with either of these promoters failed to give the desired product.Since the initial experiments of Helferich and co-workers, the scope ofthe Helferich reaction has been further extended by the use of othercatalysts and by conducting the reaction in organic solvents. Indeed,ribofuranosylation of aromatic compounds has been reported usingperacylated, β-D-ribofuranoses in organic solvents with borontrifluoride diethyl etherate as the catalyst [L. Kalvoda, Coll. Czech.Chem. Comm., 38, 1679, (1973) and the PCT application based on0125528.0]. However, substitution of X—OH for the aromatic compoundsused in these examples gave no riboside. Additional Helferich reactionswere attempted in acetonitrile, chloroform, dichloromethane,1,2-dichloroethane, diethyl ether, and nitromethane with eitherβ-D-ribofuranose tetraacetate, 1-O-acetyl-β-D-ribofuranose tribenzoateor 1-p-nitrobenzoyl-β-D-ribofuranose tribenzoate and the following Lewisacid catalysts; aluminium trichloride, boron trifluoride diethyletherate, iron(III)chloride, tin(IV)chloride, TMS-triflate and PTSA. Noreaction was successful.

Thioglycosides have proved versatile as glycosyl donors [K. Toshima andK. Tatsuta, loc. cit. ]. A reagent considered suitable for ribosylation,phenylthio β-D-ribofuranoside, was conveniently prepared fromβ-D-ribofuranose tetraacetate and thiophenol [G. Kim et al., Tetr.Lettr. 34, 7627, (1993)]. Treatment of this thioglycoside and X—OH witheither mercury(II)chloride or mercury(II)sulfate as the promoter indichloromethane or nitromethane failed to induce any riboside formation.Crich and Smith have recently developed a glycosylation procedure basedon thioglycosides that is both mild and has been applied to a range ofaglycones [D. Crich and M. Smith, J. Amer. Chem. Soc., 123, 9015,(2001)]. The Crich glycosylation involves treating the appropriatethioglycoside and glycosyl acceptor with 1-benzenesulfinyl piperidine(BSP) and trifluoromethanesulfonic anhydride (Tf₂O) in dichloromethaneat low temperature (−60° C.), either with or without the hindered base2,4,6-tri-tert-butylpyrimidine (TTBP). When these conditions wereapplied to phenylthio β-D-ribofuranoside and XOH, no reaction wasobserved, neither with nor without the presence of TTBP.

Yet another access to glycosides not involving a 1-halide intermediatein the key glycosylation stage is the trichloroacetimidate method ofSchmidt [R. R. Schmidt, Angew. Chem. Int. Ed. Engl., 25, 212 (1986)].Ribofuranosyl trichloroacetimidates have been utilised as ribofuranosyldonors in glycosides syntheses [I. Chiu Machado et al., J. Carbohydr.Chem., 13, 465, (1994)]. The most suitable donor for coupling with X—OHwould be a peracylated ribofuranosyl trichloroacetimidate. This isbecause all the acyl groups on the protected-coupled product could beconveniently removed in one step by the Zemplén procedure as describedfor other indoxyl-glycosides. The synthesis of two peracylatedribofuranosyl trichloroacetimidates has been described by Verez-Bencomoand co-workers [I. Chiu-Machado et al., J. Carbohydr. Chem., 14, 551,(1995)]. Peracetylated D-ribofuranosyl trichloroacetimidate, obtained asan anomeric mixture, was successfully used by them to produceβ-D-ribofuranosides, although they did not report its use with aromaticaglycones. The starting point of their synthesis of this ribosyl donorwas methyl 2,3,5-tri-O-acetyl-β-D-ribofuranoside. This is prepared byacetylation of methyl β-D-ribofuranoside with acetic anhydride [S. J.Angyal et al., Carbohdyr. Res., 157, 83, (1986)]. Methylβ-D-ribofuranoside is itself prepared conveniently from ribose [R.Barker and H. G. Fletcher Jr, J. Org. Chem., 26, 4605, (1961)]. Reactionof the peracetylated D-ribofuranosyl trichloroacetimidate with XOH indichloromethane at ambient temperature using TMS-triflate as thecatalyst afforded new material which was UV active on tic. Separation ofthis material from other compounds by column chromatography on silicagel C60 followed by work-up resulted in the isolation of the requisiteX-β-D-ribofuranoside peracetate essentially free from any significantimpurities. De-acetylation with a catalytic quantity of sodium methoxidein methanol, followed by evaporation of the solvent and trituration ofthe solid gave X-β-D-ribofuranoside as a solid with a purity by HPLC ofaround 96%. [HPLC conditions: Column; Chromosphere 250 mm×4.6 mm C₁₈reverse-phase silica gel 5μ (ChromosExpress, Macclesfield, UK); solvent;methanol/water 1:1 v/v, 1 ml/min flow; detection, UV at 290 nm; loading,1 mg of product in 1 g of solvent, 20 μl injection; run time, 30 min;retention time approximately 17 minutes].

The process described in the second aspect of this invention isgenerally carried out with step a) occurring in an organic solvent.

The process of this invention is generally carried out with the catalystof step a) being a Lewis acid.

It is preferred that the process of this invention is carried out withthe protected β-D-ribofuranosyl trichloroacetimidate being selected fromthe group consisting of 2,3,5-tri-O-acetyl-β-D-riboturanosyltrichloroacetimidate and 2,3,5-tri-O-benzoyl-β-D-ribofuranosyltrichloroacetimidate.

As described above T⁵ in formula III can be acyl, a trialkylsilylprotecting group or other protecting group. The most usefultrialkylsilyl protecting groups are trimethylsilyl,t-butyidimethylsilyl, triethylsilyl and triisopropylsilyl. The preferredprotecting group at T⁵ is acetyl or trimethylsilyl. When the protectinggroups are removed during step b) T⁵ may be converted into R⁵. This maybe the procedure followed when T⁵ is acetyl and R⁵ is H. Alternativelyno change may occur and T⁵ and R⁵ are identical. In another embodimentT⁵ may be modified in a separate step to produce R⁵. For example, afterstep a) the intermediate product may have an acetyl group at T⁵ which isselectively deacetylated and a subsequent reaction at the indoxyl-Nproduces R⁵. A further deprotection occurs, step b), before the indoxylβ-D-ribofuranoside is produced.

As described above, the third aspect of this invention provides a methodfor detecting β-D-ribofuranosidase activity in a sample.

In this aspect of the invention there may also be a preliminary step inwhich the sample which contains cells, usually microbial cells, is grownin or on a medium.

In an alternative embodiment the method may also comprise detecting asecond enzyme activity in the sample and in step a) the sample isadditionally contacted with a second enzyme substrate comprising anenzyme cleavable moiety which is not β-D-ribofuranosyl and an indicatormoiety which is not identical to that of the indoxyl β-D-ribofuranoside,said enzyme cleavable moiety being cleavable by the second enzymeactivity releasing the indicator moiety which forms a second detectablecompound; and

c) concluding whether the second enzyme activity is present in thesample by detecting whether. a second detectable compound is formed fromthe indicator moiety of the second enzyme substrate.

The second enzyme substrate comprises an indicator moiety which isgenerally a chromogenic moiety, but can also be a fluorogenic moiety. Itis preferred that the second enzyme substrate comprises an enzymecleavable moiety which is a sugar moiety or a phosphate or an ester suchas a caprylate.

In the method of the third aspect of this invention the sample maycomprise living cells such as bacteria or other prokaryotes, fungi,eukaryotic organisms, cell cultures or alternatively cell extracts.

It is preferred that the detection methods of this invention occur withthe sample on a solid medium and the coloured compound or compoundsproduced are insoluble.

In a specially preferred embodiment of this invention the method of thethird aspect of this invention comprises a solid medium comprising anindoxyl β-D-ribofuranoside of the current invention.

In one embodiment of the method the coloured compound formed from theindoxyl portion of the indoxyl-β-D-ribofuranoside is visualisedsimultaneously with a coloured compound formed from a chromogenic moietyof the second enzyme substrate.

In an alternative embodiment a coloured compound formed from thechromogenic moiety of the second enzyme substrate masks the colouredcompound formed from the indoxyl moiety of the indoxylβ-D-ribofuranoside substrate.

In an alternative embodiment of the third aspect of this invention thesample is in a liquid microbial growth medium.

The fourth aspect of this invention, as described above, provides a kitfor carrying out detection using an indoxyl β-D-ribofuranoside of thecurrent invention. It is preferred that the component for use inproducing a medium is for producing a solid microbial growth medium.

According to a fifth aspect of the invention, as described above, theother component in the composition is a component of solid media.Alternatively the other component may be another chromogenic enzymesubstrate. A composition of this invention may comprise a plurality ofother components sufficient to form a detection medium useful in thisinvention.

The current invention is further illustrated by the following examples.

EXAMPLE 1 Synthesis of 5-bromo-4-chloro-3-indolyl β-D-ribofuranoside(X-β-D-ribofuranoside)

The reaction was conducted in a 500 ml two-necked flask equipped with amagnetic stirrer.

To a mixture of 2,3,5-tri-O-acetyl-α/β-D-ribofuranosyltrichloroacetimide [I. Chiu-Machado et al., (1995), loc. cit.] (145.0 g)and 3 Å molecular sieves (2.0 g) in dry dichloromethane (300 ml) wasadded dry X—OH (82.9 g) and the whole mixture was stirred at roomtemperature for 10 mins. TMS-triflate (8 ml) was then added in oneportion by syringe and the reaction was left stirring at roomtemperature for 1 hour. The mixture was then poured into dichloromethane(3 L) and washed with 1 M sodium hydroxide (4×2 L). The organic layerwas separated, filtered through celite and concentrated, in vacuo, tolow volume (approx. 1 L). The organic layer was then further washed with1 M sodium hydroxide (6×1 L) and deionised water (1 L) After drying(magnesium sulfate) it was filtered through celite and concentrated, invacuo, to afford the crude protected product as a dark brown solid (87.4g).

The dark brown solid was examined by tic and found to contain two mainproducts with R_(f) values of approximately 0.37 (the protectedβ-ribofuranoside) and 0.43. [Silica gel plates, 60/80 petroleum ether,ethyl acetate 1:1 v/v, uv at 254 nm]. Flash chromatography of the crudeproduct on Silica Gel C60 (600 g) using 60/80 petroleum ether/ethylacetate/triethylamine 1:1:0.05 v/v/v as the eluting solvent gave theprotected product as a brown oil (55 g). The oil was mainly a mixture ofthe X-β-D-ribofuranoside tetraacetate and the contaminant with R_(f)0.43. This oil was taken up in warm methanol (30 ml) and after leavingat ambient temperature a precipitate was formed. After 16 hours, thecream coloured solid consisting almost entirely of material with R_(f)value 0.43 was removed by filtration, and the filtrate concentrated at40° C. in vacuo to afford X-β-D-ribofuranoside tetraacetate as a brownoil (33.6 g).

X-β-D-ribofuranoside tetraacetate (30 g) was dissolved in methanol (200ml) and 5 ml of a solution of sodium methoxide (made from 1 g sodium in20 ml of methanol) was added dropwise until the solution reached pH10.The solution was left standing at room temperature for 90 mins. thenconcentrated, in vacuo, to a brown tarry oil. The oil was triturated inacetone (500 ml). A grey solid-precipitated and this was removed byvacuum filtration. The solid was discarded and the filtrate wasconcentrated in vacuo, to a brown oil (19.3 g). The oil was trituratedwith methanol (80 ml) and the product precipitated as a pale blue solidwhich was recovered by filtration (2.57 g). The filtrate wasconcentrated, in vacuo, and triturated with methanol (30 ml) to obtain asecond crop as a pale cream solid that was also recovered by filtration(4.04 g). Both crops were combined and washed with cold acetone (approx.20 ml) followed by filtration to recover the title compound as a whitesolid (approx. 2.5 g).

EXAMPLE 2 Comparison of the Attributes of Substrates forβ-D-ribofuranosidase

The following media were produced to demonstrate the utility of thecompounds of the current invention.

Medium A (components per liter) Columbia agar (Oxoid) 40 g5-Bromo-4-chloro-3-indolyl β-D-ribofuranoside (also known 80 mg asX-β-D-ribofuranoside, a compound of this invention) 6-chloro-3-indolylβ-D-glucopyranoside 200 mg Medium B (components per liter) Columbia agar(Oxoid) 40 g 3′4′-dihydroxyflavone-4′-β-D-ribofuranoside 300 mg6-chloro-3-indolyl β-D-glucopyranoside 200 mg Ferric ammonium citrate500 mg Medium C (components per liter) Columbia agar (Oxoid) 40 g5-Bromo-4-chloro-3-indolyl β-D-ribofuranoside 80 mg(X-β-D-ribofuranoside) 3,4-cyclohexenoesculetin-β-D-galactopyranoside300 mg Isopropyl-β-D-thiogalactopyranoside 30 mg Ferric ammonium citrate500 mg Medium D (components per liter) Columbia agar (Oxoid) 40 g5-Bromo-4-chloro-3-indolyl β-D-ribofuranoside 80 mg(X-β-D-ribofuranoside)3′4′-dihydroxy-3-methoxyflavone-4′-β-D-galactopyranoside 300 mgIsopropyl-β-D-thiogalactopyranoside 30 mg Ferric ammonium citrate 500 mgMedium E (components per liter) Columbia agar (Oxoid) 40 g5-Bromo-4-chloro-3-indolyl β-D-ribofuranoside 80 mg(X-β-D-ribofuranoside) Medium F (components per liter) Columbia agar(Oxoid) 40 g 3′4′-dihydroxyflavone-4′-β-D-ribofuranoside 300 mg Ferricammonium citrate 500 mg

The flavone derivatives used in Media B, D and F are synthesised asdescribed in copending PCT application claiming priority from GB0125532.2.

All components were added to 1 litre of deionised water and autoclavedat 116° C. for 10 minutes. Culture plates were prepared from molten agarat 50° C. and dried. Various control strains with known enzymaticcharacteristics were prepared at a suspension of approximately 10⁸cfu/ml using a densitometer 10 μl of this suspension was inoculated ontoeach of the different types of media and incubated overnight at 37° C.The cultural appearance of the various strains tested are shown in Table1.

TABLE 1 Colonial appearance of various strains on various chromogenicagars. A B C D E F Enterobacter cloacae NCTC 11936 Purple Black BlackBlack Green Black Escherichia coli NCTC 10418 Green Black Black BlackGreen Black Klebsiella pneumoniae NCTC 10896 Purple Black Black BlackGreen Black Salmonella typhimurium NCTC 74 Green Black Green Green GreenBlack Serratia marcescens NCTC 10211 Purple Black Green Black GreenBlack Yersinia enterocolitica NCTC 11176 Colourless ColourlessColourless Grey/black Colourless Colourless E Acinetobacter lwoffii ATCC15309 Colourless Aeromonas hydrophila NCTC 8049 Green Citrobacterfreundii NCTC 9750 Green Enterobacter aerogenes NCIMB 10102 GreenEscherichia coli 0157 NCTC 12079 Green Shigella boydii NCTC 9327 GreenVibrio cholerae NCTC 12945 Colourless Pseudomonas aeruginosa NCTC 10662Colourless

The above described analysis indicates that when X-β-D-ribofuranoside(5-bromo-4-chloro-3-indolyl β-D-ribofuranoside) was tested with Roseglucoside (6-chloro-3-indolyl β-D-glucopyranoside) strains whichproduced both β-D-ribofuranosidase and β-glucosidase produced a mixtureof colors due to hydrolysis of both chromogenic substrates (see resultsfor medium A). Such colonies were clearly distinguishable from thosewhich hydrolysed only one or neither of the two substrates.

This attribute of X-β-D-ribofuranoside is not shown by3′4′-dihydroxyflavone-4′-β-D-ribofuranoside as the iron chalet of3′,4′-dihydroxyflavone masked the presence of any β-glucosidase activity(see results for medium B). Just as this glucosidase reaction may bemasked, the hydrolysis of X-β-D-ribofuranoside itself may be masked byother chelating chromogens. For example, the results from media C and Dshow that the hydrolysis of X-β-D-ribofuranoside may be masked wheneither 3,4-cyclohexenoesculetin-β-D-galactopyranoside or3′4′-dihydroxy-3-methoxyflavone-4′-β-D-galactopyranoside are present inthe medium.

The results demonstrated above for X-β-D-ribofuranoside demonstrate thethree advantages of the current invention. Firstly the indoxylβ-D-ribofuranosides of this invention have the ability to detectβ-D-ribofuranosidase activity with high sensitivity and low substrateconcentrations. Secondly, the indoxyl β-D-ribofuranosides of thisinvention can be used simultaneously with other indoxyl substrates toproduce a medium on which a plurality of colours may be generated.

Thirdly the indoxyl β-D-ribofuranosides of this invention may also beused simultaneously with chelation based substrates which produce anindicator capable of masking the coloured precipitate generated from theindoxyl portions following β-D-ribofuranosidase activity.

1. An indoxyl β-D-ribofuranoside of the Formula II

wherein R¹⁻⁴ are independently selected from the group consisting of H,halide, nitro and C₁₋₆ alkyl; and R⁵ is selected from the groupconsisting of H, C₁₋₆ alkyl and aralkyl; or a substituted derivative ofthe indoxyl β-D-ribofuranoside.
 2. An indoxyl compound according toclaim 1 wherein the group

is selected from the group consisting of 5-bromo-4-chloro-3-indolyl,5-bromo-6-chloro-3-indolyl, 5-bromo-3-indolyl, 6-chloro-3-indolyl,3-indolyl, 4-chloro-3-indolyl, 6-bromo-3-indolyl, 6-fluoro-3-indolyl,5,7-dibromo-3-indolyl, 4,5-dichloro-3-indolyl, 5-nitro-3-indolyl,1-methyl-3-indolyl, 5-bromo-4-chloro-1-methyl-3-indolyl,5-bromo-6-chloro-1-methyl-3-indolyl, 5-bromo-1-methyl-3-indolyl,6-chloro-1-methyl-3-indolyl, 4-chloro-1-methyl-3-indolyl,6-bromo-1-methyl-3-indolyl, 6-fluoro-1-methyl-3-indolyl,5,7-dibromo-1-methyl-3-indolyl, 5,6-dibromo-3-indolyl,5,6-dibromo-1-methyl-3-indolyl and 5-nitro-1-methyl-3-indolyl and4,5-dichloro-1-methyl-3-indolyl.
 3. An indoxyl compound according toclaim 2 wherein said group is 5-bromo-4-chloro-3-indolyl.
 4. A processfor producing an indoxyl β-D-ribofuranoside according to claim 1comprising; a) contacting a protected-β-D-ribofuranosyltrichloroacetimidate with a compound of formula (III)

wherein R¹⁻⁴ are independently selected from the group consisting of H,halide, nitro and C₁₋₆ alkyl and T⁵ is selected from the groupconsisting of acyl, trialkylsilyl and other protecting groups in thepresence of a catalyst to form a protected indoxyl-β-D-ribofuranoside;and b) removing the protecting groups.
 5. A process according to claim 4wherein T⁵ is an acetyl or a trimethylsilyl group.
 6. A processaccording to claim 4 which additionally comprises intermediate stepsbetween step a) and step b) to convert T⁵ to R⁵.
 7. A process accordingto claim 4 wherein the protected β-D-ribofuranosyl trichloroacetimidateis selected from the group consisting of2,3,5-tri-O-acetyl-β-D-ribofuranosyl trichloroacetimidate and2,3,5-tri-O-benzoyl-β-D-ribofuranosyl trichloroacetimidate.
 8. Acomposition comprising an indoxyl β-D-ribofuranoside according to claim1 and another component.
 9. A composition according to claim 8 whereinthe other component is a component of a solid microbial medium.
 10. Acomposition according to claim 8 wherein the other component is anotherenzyme substrate.
 11. A composition according to claim 10 in which thesecond enzyme substrate is selected from 6-chloro-3-indolylβ-D-glucopyranoside, 3,4-cyclohexeno esculetin-β-D-galactopyranoside,and 3′,4′-dihydroxy-3-methoxy flavone-4′-β-D-galacto pyranoside.